CellCousin2: An Optimized System for Partial Ablation and Tracing of Regenerative Lineages
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PROVIDER: PRJNA1310829 | ENA |
REPOSITORIES: ENA
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Project description:Regeneration can rely on multiple cellular sources, including stem cells, self-duplicating cells, and transdifferentiating cells. A central question in regenerative biology is how these distinct lineages contribute to repair and interact within a functional regenerate. We previously developed the CellCousin system to study hcellular plasticity using inducible recombination and nitroreductase-mediated ablation in zebrafish. Here, we present CellCousin2, which introduces two key improvements for long-term tracking of spared and regenerating cells. First, to reduce background recombination, we developed a Dihydrofolate Reductase (DHFR)-CreER system with dual control: DHFR- mediated degradation in the absence of trimethoprim, and tamoxifen-dependent activation. This combination minimizes leakiness while maintaining high recombination efficiency. Second, we replaced the original nitroreductase with NTR2.0, enabling effective ablation with tenfold lower metronidazole concentration, reducing off-target effects on the liver. Together, these enhancements make CellCousin2 a robust platform for dissecting the dynamics and interactions of regenerative lineages.
2025-08-26 | GSE306445 | GEO
Project description:Cells in the prostate are postulated to have stem cell properties based on organ regeneration following castration. Through single cell RNA-seq (scRNA-Seq) analysis, we identify a rare luminal cell population in the mouse prostate that expresses stem-like markers (Sca1+, Psca+), as well as a larger population of more differentiated cells (Nkx3.1+, Pbsn+, CD133/Prom1+). Unexpectedly, both populations acquire enhanced organoid regeneration potential following castration and contribute equipotently to prostatic regeneration, as revealed by lineage tracing. Regeneration is mediated, in part, by androgen-driven expression of Nrg2, Igf1, Fgf10 and Rspo3 by distinct populations of mesenchymal cells acting in a paracrine fashion on luminal cells. Human prostate tissue contains similar differentiated and stem-like luminal subpopulations which also, collectively. acquire enhanced regenerative potential after androgen ablation therapy. Thus, nearly all luminal cells that persist post-castration contribute to prostate regeneration, not just rare stem cells.
2020-05-01 | GSE146811 | GEO
Project description:Differentiated prostate luminal cells acquire enhanced regenerative potential after androgen ablation
| PRJNA625051 | ENA
Project description:Quiescent Müller glia heterogeneity influences regenerative response following photoreceptor ablation in the zebrafish retina
| PRJNA902356 | ENA
Project description:Retinal Müller glia (MG) can act as stem-like cells to generate new neurons in both zebrafish in mice. In zebrafish, retinal regeneration is innate and robust, resulting in the replacement of lost neurons and restoration of visual function. In mice, exogenous stimulation of MG is required to reveal a dormant and, to date, limited regenerative capacity. Zebrafish studies have been key in revealing factors that promote regenerative responses in the mammalian eye. Increased understanding of how the regenerative potential of MG is regulated in zebrafish may therefore aid efforts to promote retinal repair therapeutically. Developmental signaling pathways are known to coordinate regeneration following widespread retinal cell loss. In contrast, less is known about how regeneration is regulated in the context of retinal degenerative disease, i.e., following the loss of specific retinal cell types. To address this knowledge gap, we compared transcriptomic changes between two selective cell ablation paradigms, targeted loss of rod photoreceptors or bipolar cells. Our study spanned twelve time points encompassing the entire degenerative and regenerative process of each targeted cell type. 2,531 differentially expressed genes (DEGs) were identified. Interestingly, the majority of DEGs were paradigm specific, including during MG activation phases, suggesting the nature of the injury/cell loss informs the regenerative process from initiation onward. For example, early modulation of Notch signaling was implicated in the rod but not bipolar cell ablation paradigm. Components of JAK/STAT signaling were activated in both paradigms but were more strongly induced during rod cell regeneration. We functionally validated the role of JAK/STAT signaling during rod cell regeneration using CRISPR/Cas9-based knockdown of stat3 which inhibited both MG proliferation and rod cell regeneration kinetics. Additionally, although more than a third of all DEGs are implicated as immune-system regulators, individual immune-related factors were largely paradigm specific. These data support emerging evidence that discrete “fate-biased” regenerative processes follow from selective retinal cell loss. Microarrays were used to follow gene expression changes in the eyes of fish following either rod or bipolar specific induced death.
2023-06-13 | GSE234646 | GEO
Project description:Background: The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. Methods: We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP + cells for RNAseq analysis. Results: In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPCs). RNASeq of GFAP::EGFP cells has allowed us to clearly demonstrate that indeed these cells are NSPCs. On the contrary, the GFAP::EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPCs, and we found that are mainly fated to form neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPCs cells are necessary for functional regeneration of the spinal cord. Conclusions: The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPCs, which massively proliferate to reconstitute the spinal cord, and are differentiated into neurons. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals.
2021-01-05 | GSE164204 | GEO
Project description:Human fetal progenitor tenocytes (hFPT) produced in defined cell bank systems have recently been characterized and qualified as potential therapeutic cell sources in tendon regenerative medicine. In view of further optimizing the manufacture of the cell-based active substance, the effects of hypoxic (i.e., 2% O2 partial pressure) in vitro culture expansion on key cellular characteristics or process parameters were evaluated. To this end, multiple aspects were comparatively assessed, in either normoxic incubation (i.e., 5% CO2 and 21% O2, standard conditions) or in hypoxic incubation (i.e., 5% CO2 and 2% O2, optimized conditions). Experimentally investigated parameters and endpoints comprised cellular proliferation, cellular morphology and size distribution, cell surface marker panels, cell susceptibility toward adipogenic and osteogenic induction, while relative protein expression levels were analyzed by quantitative mass spectrometry. Results outlined conserved basic cellular characteristics (i.e., surface marker panels, phenotype under chemical induction) as well as modified key cellular parameters (i.e., cell size distribution, endpoint cell yields, matrix protein contents) potentially procuring tangible benefits with regard to an optimized cell manufacturing workflow. Specific proteomic analyses further shed some light on the effects of hypoxia at a protein level, potentially orienting the further processing of hFPT for cell-free active substance manufacture. Overall, this study indicated that hypoxic incubation impacts specific hFPT key properties and enables optimized manufacture of tenocyte-based active substances in homologous standardized transplant products.
2022-02-15 | PXD028359 | Pride
Project description:Transcriptomic comparison of two selective retinal cell ablation paradigms in zebrafish reveals shared and cell-specific regenerative responses
| PRJNA982182 | ENA
Project description:In the past decades, the paradigm of three germ layers formed by gastrulation has been modified by data suggesting an existence of neuromesodermal progenitors (NMPs) that arise during gastrulation and contribute to both spinal cord and adjacent paraxial mesoderm1-5. However, there lacks direct genetic lineage tracing evidence and functional assessment of NMPs in vivo. Here, we develop a dual recombinases-mediated genetic system to specifically trace and genetically ablate Brachyury+Sox2+ NMPs. Genetic lineage tracing results and single-cell RNA sequencing analysis show that NMPs contain three distinct uni-potent and bi-potent progenitor populations for progressive differentiation into neural and mesodermal fates. Genetic ablation of NMPs by diphtheria toxin reveals a critical role of NMPs in tail formation. This study provides in vivo genetic evidence for heterogeneity of NMPs in their cell fate determination and their functional role in developing embryos.
2023-04-11 | GSE229103 | GEO
Project description:Loss of functional β-cells is a pivotal event in the dysregulation of glucose homeostasis observed in diabetes. While the plasticity of β-cells in response to increased metabolic demand has been extensively studied, the common drivers of β-cell proliferation and the molecular mechanisms governing regeneration in humans remain elusive. Here, we investigated the proliferative response following targeted chemical ablation of β-cells using diphtheria toxin (DT) and streptozotocin (STZ) in mice. Hemizygous expression of the RIP-DTR transgene enabled efficient partial ablation of β-cells, leading to close to 50% β-cell loss five days post ablation, stabilizing at this level up to 10 weeks post ablation. Transcriptomic analysis revealed a dynamic molecular landscape following ablation, with early activation of tissue remodeling and clearance of dead cells, followed by transient activation of regenerative signaling pathways. Furthermore, we observed a distinct proliferative boost in the immediate aftermath of ablation, characterized by upregulation of cell cycle progression markers. Interestingly, long-term follow-up revealed compound stress from partial β-cell loss and high-fat diet feeding induced a robust proliferative response, albeit with a negative impact on β-cell signature. Additionally, single low-dose STZ injections induced partial β-cell loss and compensatory proliferation, with the regulatory landscape deregulated by STZ injection in the long term. The compound stress of high-fat diet and STZ ablation further altered the islet transcriptional landscape, particularly affecting metabolic pathways. Overall, our study provides insights into the dynamic response of β-cells to partial ablation and highlights the impact of compound stressors on islet function and metabolism, shedding light on potential strategies for promoting β-cell regeneration in diabetes. Loss of functional β-cells is a pivotal event in the dysregulation of glucose homeostasis observed in diabetes. While the plasticity of β-cells in response to increased metabolic demand has been extensively studied, the common drivers of β-cell proliferation and the molecular mechanisms governing regeneration in humans remain elusive. Here, we investigated the proliferative response following targeted chemical ablation of β-cells using diphtheria toxin (DT) and streptozotocin (STZ) in mice. Hemizygous expression of the RIP-DTR transgene enabled efficient partial ablation of β-cells, leading to close to 50% β-cell loss five days post ablation, stabilizing at this level up to 10 weeks post ablation. Transcriptomic analysis revealed a dynamic molecular landscape following ablation, with early activation of tissue remodeling and clearance of dead cells, followed by transient activation of regenerative signaling pathways. Furthermore, we observed a distinct proliferative boost in the immediate aftermath of ablation, characterized by upregulation of cell cycle progression markers. Interestingly, long-term follow-up revealed compound stress from partial β-cell loss and high-fat diet feeding induced a robust proliferative response, albeit with a negative impact on β-cell signature. Additionally, single low-dose STZ injections induced partial β-cell loss and compensatory proliferation, with the regulatory landscape deregulated by STZ injection in the long term. The compound stress of high-fat diet and STZ ablation further altered the islet transcriptional landscape, particularly affecting metabolic pathways. Overall, our study provides insights into the dynamic response of β-cells to partial ablation and highlights the impact of compound stressors on islet function and metabolism, shedding light on potential strategies for promoting β-cell regeneration in diabetes. Loss of functional β-cells is a pivotal event in the dysregulation of glucose homeostasis observed in diabetes. While the plasticity of β-cells in response to increased metabolic demand has been extensively studied, the common drivers of β-cell proliferation and the molecular mechanisms governing regeneration in humans remain elusive. Here, we investigated the proliferative response following targeted chemical ablation of β-cells using diphtheria toxin (DT) and streptozotocin (STZ) in mice. Hemizygous expression of the RIP-DTR transgene enabled efficient partial ablation of β-cells, leading to close to 50% β-cell loss five days post ablation, stabilizing at this level up to 10 weeks post ablation. Transcriptomic analysis revealed a dynamic molecular landscape following ablation, with early activation of tissue remodeling and clearance of dead cells, followed by transient activation of regenerative signaling pathways. Furthermore, we observed a distinct proliferative boost in the immediate aftermath of ablation, characterized by upregulation of cell cycle progression markers. Interestingly, long-term follow-up revealed compound stress from partial β-cell loss and high-fat diet feeding induced a robust proliferative response, albeit with a negative impact on β-cell signature. Additionally, single low-dose STZ injections induced partial β-cell loss and compensatory proliferation, with the regulatory landscape deregulated by STZ injection in the long term. The compound stress of high-fat diet and STZ ablation further altered the islet transcriptional landscape, particularly affecting metabolic pathways. Overall, our study provides insights into the dynamic response of β-cells to partial ablation and highlights the impact of compound stressors on islet function and metabolism, shedding light on potential strategies for promoting β-cell regeneration in diabetes.
2025-02-28 | GSE256517 | GEO